Process for the preparation of pseudomonic acid a antibiotic by microbiological method

ABSTRACT

A procedure for the preparation of pseudomonic acid A comprising submerged cultivation of a Pseudomonas bacterium strain capable of biosynthesis of the substantially pure pseudomonic acid A in aerated conditions via fermentation; and isolation of the desired compound is disclosed. In particular, the procedure of the present invention comprises cultivation of the Pseudomonas sp. bacterium strain No. 19/26 deposited under accession No. NCAIM(P)B 001235 in the National Collection of the Agricultural and Industrial Microorganisms, Budapest, Hungary, or its pseudomonic acid A-producing mutant or variant, on a medium at a temperature of between about 20° C. and 30° C. containing organic nitrogen and carbon sources and, optionally, mineral salts.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of U.S. Ser. No. 09/497,807, filed Feb.3, 2000 which claims benefit of Provisional Applns No. 60/118,457 filedFeb. 3, 1999 and No. 60/137,334 filed Jun. 3, 1999.

FIELD OF THE INVENTION

The present invention relates to a microbiological method for themanufacture of the antibiotic pseudomonic acid A (mupirocin).

BACKGROUND OF THE INVENTION

Pseudomonic acid A, also known as mupirocin, is an antibiotic that has agrowth inhibiting effect mainly against Gram positive bacteria (e.g.Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae,Klebsiella pneumoniae) and some Gram negative bacteria (e.g. Haemophilusinfluenzae, Neisseria gonorrhoeae) [A. Ward, D. M. Campoli-Richards,Drugs 32, 425-444 (1986)] and its minimal inhibiting concentration is inthe range of 0.02-0.5 mg/dm³. Pseudomonic acid A, by inhibiting theisoleucine-tRNA synthase enzyme, affects the peptide synthesis ofpathogen bacteria [J. Hughes and G. Mellows, Biochem. J. 191, 209-219(1980)]. An advantageous feature of this antibiotic is that it has verylow toxicity both for humans and animals and it is negative in the Amestest. Pseudomonic acid A is presently used in human therapy, in variousformulations, for the treatment of skin infections (e.g. impetigo,pyoderma), nose and external ear infections, acne, burns, eczema,psoriasis, in case of ulceration for treatment of secondary infections,and for prevention of hospital infections.

The chemical structure of pseudomonic acid A was determined to be9-{4[5S(2S,3S-epoxy-5S-hydroxy-4S-methylhexyl)-3R,4R-dihydroxy-tetrahydropyran-2S-yl]-3-methylbut-2(E)-enoyloxy}nonanoicacid [E. B. Chain and G. Mellows, J. C. S. Chem. Comm. 847-848 (1974);R. G. Alexander, J. P. Clayton, K. Luk, N. H. Rogers, T. J. King, J. C.S. Perkin I. 561-565 (1978)], as depicted by formula (I):

It is known that Pseudomonas fluorescens is able to produce thepseudomonic acid A. According to the British Patent No. 1,395,907, thePseudomonas fluorescens NCIB 10586 strain is able to biosynthesize thepseudomonic acid complex consisting of pseudomonic acid A and its isomerbeing a double bond in the cis position between the carbon atoms C₂ andC₃ and pseudomonic acid B. The ratio of the components is 4.5:4.5:1.According to the Japanese patent application No. 52-70083, however, thePseudomonas fluorescens Y-11633 strain is able to biosynthesize thepseudomonic acid complex consisting of the pseudomonic acid A,pseudomonic acid B and further two components with unknown structures inthe ratio of 9:0.5:0.5.

SUMMARY OF THE INVENTION

The present invention is directed to a procedure for the preparation ofpseudomonic acid A comprising cultivating on a medium comprising atleast one organic nitrogen or carbon source, in submerged aeratedconditions, a Pseudomonas bacterium strain capable of the biosynthesisof pseudomonic acid A, and fermentation of the Pseudomonas culture suchthat pseudomonic acid A is formed. Preferably the Pseudomonas bacteriumstrain is Pseudomonas sp. bacterium strain No. 19/26 deposited underaccession No. NCAIM(P)B 001235 in the National Collection ofAgricultural and Industrial Microorganisms, Budapest, Hungary, or itspseudomonic acid A-producing mutant or variant.

The present invention also is directed to a Pseudomonas culture capableof biosynthesizing pseudomonic acid A in submerged aerated conditions,consisting essentially of a novel Pseudomonas sp. bacterium strain No.19/26.

The present invention further is directed to a biologically pure cultureof a novel Pseudomonas sp. bacterium strain No. 19/26.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an HPLC chromatogram of the pseudomonic acid complex producedby Pseudomonas sp. No. 19/26 [NCAIM(P)B 001235].

DETAILED DESCRIPTION OF THE INVENTION

In the course of searching antimicrobial antibiotics produced bybacteria, 20,000 microorganisms were isolated on nutrient agar mediumcontaining 10 μg/ml candicin and 5 μg/ml cycloheximide in order toprevent the growth of fungi.

Antibacterial effectiveness of the shaken flask culture of the isolatedbacterial strains was examined on different Gram positive and Gramnegative test-microorganisms and their resistant variants forantibiotics occurring in the therapeutic practice. By the application ofthe antibiotic resistant test-microorganisms we wanted to promote therecognition of antibacterial antibiotics with new mode of action.

In the course of the above described examinations a bacterium isolate,designated as “strain No. 19/26,” was selected. Originally, thisorganism had been obtained from a soil sample collected in Argentina.Its culture broth had significant antibiotic effect against Bacillussubtilis ATCC 6633, Staphylococcus aureus SMITH, thepenicillin-resistant Staphylococcus aureus 1110, an unnumberedStreptococcus pneumoniae strain, Streptococcus pyogenes A 115 ROBB,Alcaligenes faecalis 140001, Bordetella bronchiseptica ATCC 4617,Klebsiella pneumoniae ATCC 10031, the meticillin- andaminoglucoside-resistant Staphylococcus aureus MRSA 119OR and twounnumbered Mycoplasma pneumoniae and Haemophilus influenzae strains,respectively. Later the antibacterial metabolic product of strain No.19/26 was isolated from its culture broth and as a result of a chemicalstructure analysis it was found to be identical to the pseudomonic acidA.

At the same time this selected pseudomonic acid A-producing bacteriumstrain was subjected to taxonomic studies. First a preliminarycomparative investigation was done with selected closely relatedbacterial strains using the Bio-Merieux ATB Expression equipment with ID32 GN strip. The latter is suitable for a relatively fast (e.g. genericlevel) taxonomic identification of a given unknown bacterial strain ifits diagnostic properties can show a desired high degree of pheneticsimilarity to at least one of those of the authentic strains that wereincorporated in the database of the equipment. Carbon-source utilizationspectra of an unknown bacterial strain to be investigated can bedetermined with this equipment in 32 tests with 14 different sugars and14 organic acids, respectively, added into a minimal culture mediumcompleted with a complex of growth factors. Evaluation of theutilization test results is done after the inoculation and passing 48hours incubation time by means of automatic turbidity measurement on thedeveloped microcultures designating the growth intensities (thereactions) as + or − or ?, respectively. The results of thesynchronously studied biochemical tests are evaluated by the software ofthe equipment. The obtained physiological-biochemical data of strain No.19/26 were correlated with those of the bacterial strains that arelisted in the database (the total number of such strains is in this case114, among them 14 belonging to different Pseudomonas species:Pseudomonas aeruginosa I and II, Pseudomonas alcaligenes, Pseudomonasfluorescens I and II, Pseudomonas mendocina, Pseudomonas mesophilica,Pseudomonas pickettii, Pseudomonas pseudomallei, Pseudomonasvesicularis, Pseudomonas cepacia, Pseudomonas diminuta, Pseudomonasputida, and Pseudomonas stutzeri). According to the outcome of thiscorrelation experiment, strain No. 19/26 proved to be a member of thegenus Pseudomonas.

After successful identification at generic level of strains No. 19/26 wetried to identify it at species level by means of classical taxonomicinvestigation method.

Cultures of strain No. 19/26 can multiply on suitable solid media inform of non-sporulating Gram negative rods, 0.6-1.1 to 1.3-4.0 microns.These are actively motile with polar flagella. These obligately aerobicchemoorganotrophic rods can attack glucose only oxidatively. They cannotferment and are unable to respire with nitrates as terminal electronacceptors, but show positive catalase reaction. They do not developvisible colonies on simple synthetic media and for their multiplicationalways require growth factors. Growing under optimal conditions theircolonies are arginindihydrolase positive, produce fluorescent pigmentsbut the presence and accumulation of poly-β-hydroxy-butyrate in thecells cannot be detected.

Further features of strain No. 19/26 are the following: development ofits colonies was not observed at 41° C.; it does not produce levan fromsucrose, it does not hydrolyze gelatin and starch, it is oxidasepositive, and it does not utilize glucose, 2-keto-gluconate, trehaloseand meso-inositol as the sole carbon source.

All of these diagnostic properties clearly separate the Pseudomonasstrain No. 19/26 from the species P. aeruginosa, P. fluorescens, P.chlororaphis, P. aureofaciens, P. syringae, P. viridiflava, P. cichorii,P. stutzeri, P. mendocina, P. alcaligenes, P. pseudoalcaligenes and P.putida species. Cultivation of strain No. 19/26 can be carried out onlyon media prepared with (0.2-1.0%) yeast extracts, corn steep liquor orother complex natural nutrients containing very different growthfactors.

On the basis of these particular diagnostic properties strain No. 19/26can be considered as an organism which belongs to the so called“non-typical Pseudomonas species.” Such “non-typical Pseudomonasspecies” may be incorporated into a newly established bacterial taxon inthe future. (N. J. Palleroni, “Psuedomonaceae,” Bergey's Manual ofSystematic Bacteriology, 1,141-219, ed.: N. R. Krieg and J. G. Holt,Baltimore: Williams and Wilkins, 1984)

Strain No. 19/26 was deposited on Jul. 16, 1996 under accession No.NCAIM(P)B 001235 in the National Collection of Agricultural andIndustrial Microorganisms, Budapest, Hungary.

According to a preferred method of the invention the Pseudomonas sp.,bacterium strain No. 19/26 deposited under accession No. NCAIM(P)B001235 is used for the manufacture of substantially pure pseudomonicacid A. The selected strain can use peptone, beef extract, corn steepliquor, yeast extract, casein and soy-bean meal as nitrogen sources.Besides the above nitrogen sources, carbon sources such as glucose,glycerol and sunflower oil can be applied in different combinations.

Although the medium components of natural origin contain mineral salts,it is advantageous to add some mineral salts (e.g. ammonium, calcium,iron, zinc, copper, magnesium, manganese, sodium or potassium salts) tothe seed culture medium. Magnesium sulfate, manganese dichloride,ferrous sulfate, zinc chloride, copper II sulfate, ammonium sulfate,potassium dihydrogen phosphate, sodium chloride and calcium carbonateare preferred.

Maintenance of the strain is carried out on slant agar medium containingpeptone-casein at 4° C., freshly transferring it every two weeks. Tomaintain the pseudomonic acid A productivity, the strain can be storedproperly both by deep-freezing the culture or in lyophilized form.

In the course of the fermentation, the Pseudomonas sp. No. 19/26 strainis seeded into a suitable medium and cultivated in submerged and aeratedfermentation conditions. The pH of the culture medium is preferably setto a neutral value (pH=about 7.0), the temperature of the cultivationbetween is about 20° C. and about 30° C., preferably between about 24°C. and about 26° C. Depending on the fermentation conditions the maximumvalue of the antibiotic productivity can be reached after 50-60 hours.The pseudomonic acid complex being formed in the course of thefermentation comprises substantially pure pseudomonic acid A, althoughas a result of the biosynthesis, small quantities of the pseudomonicacid B of formula (II)

and pseudomonic acid C of formula (III)

are also formed.

The antibacterial activity of the pseudomonic acid antibiotic complex isdetermined by agar diffusion microbial method. The medium is abeef-extract-peptone-glucose-containing agar, its pH is 6.5 and the testorganism is Bacillus subtilis ATCC 6633. The activity value obtained bythe microbiological method represents primarily the quantity of thepseudomonic acid A, since the quantity of the other pseudomonic acidcomponents is very small, and relating to the component A their specificactivity against the test-microorganism—especially in the case of thecomponent B—is considerably weaker.

In the course of the fermentation the exact quantity of the pseudomonicacid A and the accompanying minor components in the fermentation brothare determined by high pressure liquid chromatography (HPLC) in whichthe supernatant of the ultrasonic treated and centrifuged sample of thebroth diluted to twice by ethanol is investigated (equipment: LKB 2248pump, LKB 2141 UV detector (analysis at 222 nm), column: Nucleosil C₈ 10μm (BST), eluent: mixture (35:65) of acetonitrile and 0.1 M NH₄H₂PO₄solution (pH:5.0), flow rate: 1.2 ml/min), Retention times: pseudomonicacid A (PSA) is 8.5 min, pseudomonic acid B (PSB) is 6.0 min, andpseudomonic acid C (PSC) is 22.5 min.

The HPLC chromatogram of the pseudomonic acid antibiotic complexbiosynthesized by Pseudomonas sp. No. 19/26 strain can be seen in FIG.1. This chromatogram indicates that the pseudomonic acid complexobtained by the fermentation acid No. 19/26 strain of substantially purepseudomonic acid A. The total quantity of the pseudomonic acid B andpseudomonic acid C components is below 5%. In this way it became evidentthat the Pseudomonas sp. No. 19/26 strain is able to biosynthesize theantibiotic in a more favorable composition, than the pseudomonic acidcomplex producing—Pseudomonas fluorescens strains published earlier.This fact is advantageous for industrial production.

The process according to the present invention is illustrated by theexample below. However, the present invention should not be construed aslimited thereby.

EXAMPLE

Pseudomonas sp. No. 19/26 strain was maintained on PCA marked slant agarmedium. Composition of the PCA medium was the following:

beef extract 3 g peptone 5 g agar 15 g

diluted up to 1000 ml with distilled water. The pH of the medium was7.0-7.2.

Seeded slant agar was incubated at 25° C. for 24 hours, and the cellswere suspended in 5 ml normal saline solution (cell number in thesuspension: 10⁹-10¹⁰/ml). 1 ml of the suspension obtained was seededinto 100 ml I-21 marked seed culture medium sterilized in a 500 mlErlenmeyer flask. Composition of I-21 medium was the following:

glucose 10 g glycerol 5 g corn steep liquor 3 g ammonium sulfate 2 gpotassium dihydrogen phosphate 0.4 g magnesium sulfate-water (1:7) 0.4 gmanganese dichloride-water (1:2) 0.03 g calcium carbonate 4 g sunfloweroil 2 g

diluted up to 1000 ml with tap water. The pH of the medium is set to 7.0before sterilization.

The flask containing the seeded medium was shaken on a shaking table(260 RPM, amplitude 10 cm) at 25° C. for 18-20 hours. After this 50 ml(1%) of the shaken culture was seeded into 5 liter medium with mark E-5sterilized at 121 ° C. for 45 min in a 10 liter jar fermentor.Composition of E-5 medium was the following:

glucose* 50 g glycerol 50 g soy-bean meal 100 g corn steep liquor 15 gsodium chloride 25 g calcium carbonate 25 g sunflower oil 10 g

diluted up to 5 liters with tap water. (*Glucose was sterilized in 50%solution for 30 min, and added into the medium together with the seedingmaterial.) The pH of the medium was set to 7.0 before sterilization.

The cultivation was stirred and aerated for 50-60 hours. The temperatureof the cultivation was 25° C., stirring rate was 500 RPM, airflow ratewas 200 liters/hour. As an antifoaming agent, sunflower oil was used inminimal quantity (about 20-30 ml per fermentor).

The biosynthesis of the pseudomonic acid complex was started at hours8-10 of the fermentation and the maximal antibiotic concentration wasexperienced at hours 55-60 hours of the culture.

Isolation of the pseudomonic acid A from the fermentation broth wascarried out as follows: After finishing the fermentation, the 4.5 literculture broth obtained was centrifuged, then the pH of the supernatant(4.06 liter) was adjusted to 4.5 with diluted (20%) sulfuric acid. Theacidified liquor was then twice extracted by 2.03 liters ethyl acetate.The phases were separated and a sharp phase was prepared from theemulsive organic phase by centrifugation. The combined extract wasevaporated in vacuum. The crude product obtained (2.45 g) was dissolvedin a 25 ml mixture of chloroform-methanol-99.5% acetic acid (93:5:2),and the solution obtained was loaded on the column(height:diameter=28.5) prepared from 245 g Kieselgel 60 (particle size:0.063-0.2 mm; Reanal) and with the above solvent mixture. Elution wascarried out with the above solvent mixture. In the course of the elution50 ml fractions were collected and the pseudomonic acid A content of thefractions was analyzed by thin layer chromatography using Kieselgel 60(DC-Alufolien: 105554, Merck) adsorbent and chloroform-methanol-99.5%acetic acid (90:8:2) developing solvent mixture. Fractions 34-50 elutedfrom the Kieselgel 60 column contained pseudomonic acid A. Thesefractions were combined (850 ml) and meanwhile cooling 280 ml water wasadded to the solution obtained. After this the pH of the solutionmixture was adjusted to 4.5 with 1N aqueous sodium hydroxide solution.The organic solution was separated from the water phase, then the waterphase was extracted again by 280 ml chloroform. The combined extract wasevaporated in vacuum, thus pure pseudomonic acid A could be obtained.

The column chromatographic fractions 14-15 contained pseudomonic acid C,while fraction 54 contained pseudomonic acid B, from the minorcomponents could be recovered in pure form by the above writtenprocedure.

The spectroscopic characterization of the isolated pseudomonic acidcomponents was described using the numbering system of structuralformula (I), shown above.

Spectroscopic Characterization of the Pseudomonic Acid A

Ultraviolet spectrum (10 μg/ml, in 95% ethanol solution): λ_(max)=222 nm

E_(1 cm) ^(1%)=303.6

Infrared spectrum (KBr): ν_(OH) 3483 and 3306, ν_(c=o) 1728 (COOCH₂),1720 (COOH) cm⁻¹ ¹H-NMR spectrum (CDCl_(3,)δ_(TMS) = 0): δ [ppm],(integral), multiplicity Coupling constant (Hz) Assignment 5.75 (1H) q⁴J_(2,15) = 1.1 2-H 4.08 (2H)t ³J_(8′,9′) = 6.4 9′-H₂ 3.72-3.93 (4H)m5-H; 7-H; 13-H; 16-H_(a) 3.55 (1H) dd ²J_(16a,16b) = 11.8; ³J_(16b,8) =2.6 16-H_(b) 3.48 (1H)dd ³J_(5,6) = 8.4; ³J_(6,7) = 3.2 6-H 2.82 (1H)td³J_(9,10) = 6.3; ³J_(10,11) = 2.3 10-H 2.74 (1H)dd ³J_(10,11) = 2.3;³J_(11,12) = 7.8 11-H 2.60 (1H)dd ²J_(4a,4b) = 14.5; ³J_(4a,5) = 2.74-H_(a) 2.28-2.36 (3H)m 4-H_(b); 2′-H₂ 2.20 (3H)d ⁴J_(2,15) = 1.1 15-H₃2.02 (1H)m 8-H 1.61-1.76 (6H)m 9-H₂; 3′-H₂; 8′-H₂ 1.33-1.43 (9H)m 12-H;4′-H₂; 5′- H₂′; 6′-H₂; 7′-H₂ 1.22 (3H)d ³J_(13,14) = 6.4 14-H₃ 0.94(3H)d ³J_(12,17) = 7.0 17-H₃ ¹³C-NMR spectrum (CDCl₃ solution, δ_(TMS) =0): δ [ppm] Assignment δ [ppm] Assignment 177.8 s C-1′ 42.7t,d C-4, C-12166.9s C-1 39.4d C-8 156.0s C-3 33.9t,t C-9, C-2′ 117.7d C-2 31.6t C-4′*74.9d C-5 28.9t C-5′* 71.4d C-13 28.8t C-6′* 70.4d C-7 28.5t C-8′* 69.0dC-6 25.9t C-7′ 65.3t C-16 24.6t C-3′ 63.9t C-9′ 20.8q C-14 61.3d C-1119.1q C-15 55.6d C-10 12.7q C-17 *interchangeable assignments Chemicalionization (CI) mass spectrum: Characteristic spectral data: m/z R.I.(%) Assignment 501 100  [M + H]⁺ 327 45 [M + H—HO/CH₂/₈COOH]⁺ 309 16[m/z 327-H₂O]⁺ 227 33 [C₁₂H₁₉O₄]⁺

Spectroscopic Characterization of the Pseudomonic Acid B

Ultraviolet spectrum (10 μg/ml, in 95% ethanol solution): λ_(max)=222 nm

E_(1 cm) ^(1%)=280

Infrared spectrum (film): ν_(OH)3418, ν_(c=o)1713 (COOCH₂, COOH)cm⁻¹¹H-NMR spectrum (CDCl_(3,) δ_(TMS) = 0): δ [ppm], (integral),multiplicity Coupling constant (Hz) Assignment 5.68 (1H) s 2-H 4.02(2H)t ³J_(8′,9′) = 6.6 9′-H₂ 3.7 (2H)m 7-H,13-H 3.55 (1H)td ³J_(4a, 5) =³J_(5,6) = 5-H 9.3;³J_(4b,5) = 1.8 3.41 (2H)dd ²J_(16a,16b) = 11.0 16-H₂3.24 (1H)dd ³J_(5,6) = 9.3; ³J_(6,7) = 2.8 6-H 2.92 (1H)td ³J_(9,10) =5.6; ³J_(10,11) = 2.0 10-H 2.69 (1H)dd ³J_(10,11) = 2.0; ³J_(11,12) =7.2 11-H 2.62(1H)dd ²J_(4a,4b) = 14.5; ³J_(4a,5) = 1.9 4-H_(a) 2.20(2H)t ³J_(2′,3′) = 7.4 2′-H₂ 2.14 (3H)br.s 15-H₃ 2.10 (1H)dd ²J_(4a,4b)= 14.5; ³J_(4b,5) = 9.3 4-H_(b) 1.85 (1H)dd ²J_(9a,9b) = 14.3;³J_(9a,10) = 5.3 9-H_(a) 1.20-1.68 (14H)m 9-H_(b); 12-H; 3′-H_(2;) 4′-H₂; 5′-H₂; 6′-H₂; 7′-H₂; 8′-H₂ 1.14(3H)d ³J_(13,14) = 6.4 14-H₃ 0.88(3H)d ³J_(12,17) = 7.1 17-H₃

Chemical ionization (CI) mass spectrum:

Characteristic spectral data:

m/z RI (%) Assignment 517 100 [M + H]⁺ 343  70 [M + H—HO/CH₂/₈COOH]⁺

Spectroscopic Characterization of the Pseudomonic Acid C

Ultraviolet spectrum (10 μg/ml, in 95% ethanol solution): λ_(max)=222 nm

E_(1 cm) ^(1%)=307.

Infrared spectrum (film): ν_(OH)3455, ν_(c=o)1713 (COOCH₂, COOH)cm⁻¹¹H-NMR spectrum (CDCl_(3,) δ_(TMS) = 0): δ [ppm] (integral),multiplicity Coupling constant (Hz) Assignment 5.69 (1H)s 2-H 5.25-5.50(2H)m 10-H; 11-H 4.00 (2H)t ³J_(8′),_(9′) = 6.5 9′-H₂ 3.88 (1H)dd³J_(6,7) = ³J_(7,8) = 3.0 7-H 3.78 (1H)dd ²J_(16a,16b) = 11.8; 16-H_(a)³J_(16a,8) = 2.6 3.68 (1H)dd ³J_(4b,5) = 2.5;³J_(5,6) = 9.0 5-H 3.55(1H)qd ³J_(12,13) = ³J_(13,14) = 6.3 13-H 3.49 (1H)dd ²J_(16a,16b) =11.8; 16-H_(b) ³J_(16b,8) = 2.0 3.41 (1H)dd ³J_(5,6) = 9.0;³J_(6,7) =3.0 6-H 2.58 (1H)dd ²J_(4a,4b) = 13.8; ³J_(4a,5) = 2.5 4-H_(a) 2.20(2H)t ³J_(2′,3′) = 7.4 2′-H₂ 2.00-2.30 (4H)m 4-H_(b); 9-H₂;12-H 2.10(3H)m 15-H₃ 1.78 (1H)m 8-H 1.45-1.65 (4H)m 3′-H₂; 8′-H₂ 1.15-1.35 (8H)m4′-H₂; 5′-H₂; 6′-H₂, 7′-H₂ 1.08 (3H)d ³J_(13,14) = 6.3 14-H₃ 0.92 (3H)d³J_(12,17) = 6.8 17-H₃

Chemical ionization (CI) mass spectrum:

Characteristic spectral data:

m/z R.I. (%) Assignment 485 30 [M + H]⁺ 287 60 [M + H—HO/CH₂/₈COOH]⁺

Although certain presently preferred embodiments of the invention havebeen described herein, it will be apparent to those skilled in the artto which the invention pertains that variations and modifications of thedescribe embodiment may be made without departing from the spirit andscope of the invention. Accordingly, it is intended that the inventionbe limited only to the extent required by the appended claims and theapplicable rules of the law.

We claim:
 1. A biologically pure culture of Pseudomonas sp. bacteriumstrain No. 19/26 deposited under accession No. NCAIM(P)B 001235 in theNational Collection of Agricultural and Industrial Microorganisms,Budapest, Hungary.